The antigen-antibody reaction is the basis for all immunological test methods. Certain proteins known as antibodies are produced by mammals in response to the presence of an antigen, that is a foreign substance, which can be another protein or a carbohydrate. This normal body response to a foreign substance has led to the development of a number of techniques which are used to diagnose various diseases, disorders and physiological conditions. In a general sense, the component of the antibody-antigen reaction to be detected is defined herein as the immune species while the other corresponding component is considered the receptor.
For example, in vitro tests for the presence of a suspected protein, antigen or antibody in a biological sample are carried out by adding the immunological counterpart to the biological sample. If the suspected substance is present, the resulting antigen-antibody reaction can be demonstrated by precipitation of the antigen-antibody complex. This reaction complex is generally difficult to detect visually. For this reason, either antibodies or antigens are often bound to insoluble particles, for example polymer latex particles, so that when the complex is formed, it is readily detectable from the resulting agglutination either by observing the presence of clumping or a detectable tracer associated with the particles. Agglutination then is characterized by the clumping of particles from a suspension of particles. Further details of known agglutination methods are provided in U.S. Pat. Nos. 4,419,453 (issued Dec. 6, 1983 to Dorman et al.) and 4,459,361 (issued July 10, 1984 to Gefter).
Of the several groups of Streptococci, group A Streptococcus (S. pyogenes) is primarily responsible for causing pathological conditions in humans, such as B-hemolytic pneumonia, scarlet fever, rheumatic fever, cardiac sequelae, glomerulonephritis, septic sore throat and puerperal sepsis. Because of the serious nature of infections potentially caused by Streptococcus A, it is important to diagnose its presence in an early stage of infection so that an appropriate course of treatment may be selected. Early tests for detection required culturing a biological sample for long periods of time, usually at least 18 and up to 48 hours. In most cases, such lengthy tests delay treatment making them undesirable. Some doctors must forward test cultures to laboratories for evaluation by mail or courier, thereby delaying treatment further.
Most recent tests for Streptococcus A have been described which are allegedly quicker than the culturing techniques. U.S. Pat. No. 4,618,576 (issued Oct. 21, 1986 to Rosenstein et al.) describes an agglutination test using certain enzymes to extract the antigen directly from the swab used to obtain a specimen from the throat. A kit comprising an applicator means for collecting the specimen, an extraction reagent containing the enzymes and suitable indicator reagents is also described. The described method is disadvantageous because the agglutinate is not readily observable except with a microscope, and requires extraction enzymes prepared from a bacterium which must be cultured.
Another agglutination assay is described in E. P. Publication No. 150,567 (Meridian Diagnostics, published Aug. 7, 1985). This assay describes an extraction technique which uses a solution of sodium nitrite combined with glacial acetic acid. This assay has a serious disadvantage, however, because the extraction composition is not easily stored, transported or handled. Acetic acid is a volatile liquid at room temperature, the normal conditions of use. It therefore presents problems in long-term storage and ease of handling.
U.S. Pat. No. 4,673,639 (issued June 16, 1987 to Slifkin) allegedly describes a means for overcoming the problems associated with volatile extraction reagents. This reference proposes affixing one or both extraction reagents, in combination with a water-soluble binder material, in a ready-to-use microtube. The reagents used include a nonvolatile acid, such as citric acid. The patent also teaches a further advantage of this method over known assays in that a neutralizing step is unnecessary (see Col. 4, lines 4-18). Eliminating this step allegedly has no adverse effects on the agglutination reagents used or the assay results.
We have found that elimination of a neutralizing step adversely affects certain agglutination reagents in an assay for Streptococcus A antigen. It can also adversely affect antibodies if they are in a low pH environment too long. Furthermore, it would be desirable to eliminate the need for using a binder material to immobilize extraction reagents because of the additional material and manufacturing expense they engender.